Enhanced Patient Serum Immunoreactivity to Recombinant Mycobacterium tuberculosis CFP32 Produced in the Yeast Pichia Pastoris Compared to Escherichia coli and Its Potential for Serodiagnosis of Tuberculosis
International Journal of Infectious Diseases
CFP32 is a Mycobacterium tuberculosis complexrestricted secreted protein that was previously reported to be present in a majority of sputum samples from patients with active tuberculosis (TB) and to stimulate serum antibody production. CFP32 (annotated as Rv0577) was therefore considered a good candidate target antigen for the rapid serodiagnosis of TB. However, the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant CFP32 (rCFP32) produced
... (rCFP32) produced in Escherichia coli was used as the test antibody-capture antigen, a potential shortcoming stemming from differences in bacterial protein posttranslational modifications. To further investigate the serodiagnostic potential of rCFP32 synthesized in different heterologous hosts, we expressed rCFP32 in the yeast Pichia pastoris. Compared to E. coli rCFP32, yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies. Likewise, yeast rCFP32 was significantly better recognized by sera from TB patients, in enzyme-linked immunosorbent assay (ELISA), than E. coli rCFP32. In subsequent testing, the yeast rCFP32-based antibody-capture ELISA had a sensitivity of 85% and a specificity of 98% for the discrimination of active TB cases (n = 40) from BCG vaccinees (n = 39). The sensitivity was surprisingly high for a single-antigen TB serodiagnostic test compared to tests using E. coli-expressed antigens. Overall, the trans-production of rCFP32 in P. pastoris significantly improved the serologic detection of CFP32-specific antibodies in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases (International patent request CA 2,551,537 of July 3rd, 2007). , a total of 90898 samples including urine and non urine specimens were processed using the Aptima Combo 2 Assay (AC2) which has the ability to detect both Chlamydia trachomatis (CT) and Neisseria gonorrhoea (GC) in the same sample Results: Overall CT was detected in 6.8% of samples. CT was detected in 7.8% of urine specimens and 5.3% of genital specimens. CT was also detected in 69 tampon samples (12%), 34 Thin Prep samples (3.2%), 30 eye swabs (5.2%), 30 rectal swabs (8.7%), 14 throat swabs (1.4%) and 1 seminal fluid (1.1%). Overall GC was detected in 0.7% of samples. GC was detected in 0.6% of urines specimens and 0.45% of genital swabs. GC was detected in 50 throat swabs (5.1%), 41 rectal swabs (12%), 11 tampons (1.9%) and 3 eye swabs (0.5%). Dual infections with CT and GC were detected in 0.7% of all samples. Overall prevalence of GC by culture was 0.3%. None had a positive GC culture and a negative AC2 assay result. 21% of specimens positive for GC by AC2 did not have a formal request for GC by AC2 (GCNR: GC Not Requested). 29% GCNR GC positive specimens by AC2 did not have a simultaneous culture request. 18% GCNR GC positive specimen had concurrent CT infection Conclusions: CT and GC were detected by AC2 assay in a wide range of sample types. AC2 was more sensitive than culture for detection of GC. The ability of the assay to detect both CT and GC in one sample has emphasised the importance of testing for dual infections and enabled the un-requested GC to be detected. The data suggests that clinicians are opting for molecular-based testing for GC rather than culture-based methods.